Extracellular matrix (ECM)-degrading proteinases of the matrix metalloproteinase (MMP) gene family have been implicated in the pathogenesis of chronic disease states, such as arthritis, diabetes, cirrhosis and cancer progression. The MMPs also play a role in normal tissue development and remodeling. Endogenous protease inhibitors of several classes can regulate the ECM-degrading activities of MMPs, but the tissue inhibitors of metalloproteinases (TIMPs) are the most specific and well studied. TIMPs can also modulate cell growth, differentiation, and migration, as well as programmed cell death. These cellular effects may involve both indirect (via inhibition of MMP) and direct (MMP-independent) mechanisms. This project will examine at the level of molecular regulation how TIMPs alter cellular responses during tissue remodeling and differentiation. The hypothesis being tested is that, in addition to their ability to down-regulate MMP-dependent ECM remodeling, TIMPs selectively target cellular pathways involved in cell growth and differentiation, and together these activities of TIMPs facilitate the return of tissue homeostasis and/or tissue differentiation. In previous reports we demonstrate in vitro that TIMP-2 specifically inhibits the mitogenic response of human microvascular endothelial cells to basic fibroblast growth factor (FGF-2). We also show that TIMP-1 promotes B lymphocyte differentiation and prevents induction of apoptosis in human Burkitt's lymphoma cells in vitro. The present proposal addresses the cellular mechanisms by which TIMPs elicit specific cellular responses. The general approach uses parallel in vitro culture and in vivo animal experiments to analyze mechanisms of endothelial cell growth inhibition, angiogenesis and B cell differentiation. Human microvascular endothelial or lymphoid B cells in the presence of exogenous wild type and mutant (Ala+) TIMPs or with genetically altered TIMP levels will be studied in culture, as well as normal or transgenic mice, and analyzed for growth, differentiation, angiogenesis and immune functions. The mechanisms by which TIMPs inhibit endothelial proliferation and angiogenesis will be compared and contrasted with the mechanisms by which TIMPs induce differentiation and inhibit apoptosis in B cells. The mechanisms that mediate these two distinct sets of processes and their molecular targets on the cell surface, cytoplasm and nucleus will be elucidated. The direct effects of TIMPs on the phenotype of endothelial cells and B cells will be studied to evaluate their roles in altering proliferation or inducing differentiation. These studies address an important aspect of the biology of chronic disease, including cancer, which is how TIMPs regulate normal cell functions and how these functions are altered in disease states. The proposed studies may form the basis of intervention and therapy in cancer, not only in the late malignant stages, but also potentially in premalignant lesions.